Method for inhibiting reperfusion injury in the brain

ABSTRACT

The present invention relates to a method for inhibiting reperfusion injury in the brain. The method involve injecting via the carotid artery or jugular vein an antioxidant-loaded nanoparticle. A nanoparticle formulation containing an inert plasticizer is also provided for sustained release of an active agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/955,739, filed Sep. 30, 2004, which issued on Feb. 19, 2008 as U.S.Pat. 7,332,159. The entire disclosure of the aforesaid application isincorporated by reference in the present application.

BACKGROUND OF THE INVENTION

Stroke is a sudden loss of brain function resulting from interferencewith the blood supply to the central nervous system leading to cerebralischemia. Many factors play a role in the development of brain damageafter ischemia. Among these factors, oxidative stress has been shown toplay a central role in cerebral ischemia. Oxidative stress is ofteninitiated and propagated by overproduction of O₂ ⁻ and H₂O₂ and theirconversion to potent oxidants, such as hydroxyl radical andperioxynitrate. In general, free radical production is low, and theorganism can neutralize or metabolize the toxic effects by free radicalscavengers such as super oxide dismutase (SOD) and catalase (Fridovich(1983) Annu. Rev. Pharmacol. Toxicol. 23:239-57). Nevertheless, undersome pathophysiological conditions, there is oxygen radical accumulationthat impairs the cells, such as free radical accumulation duringcerebral ischemia (Ste-Marie, et al. (2000) Can. J. Neurol. Sci.27:152-9; Mori, et al. (1999) Brain Res. 816:350-7). Furthermore, theburst of free radical production has been demonstrated at the onset ofreperfusion after cerebral ischemia (Dirnagl, et al. (1995) J. Cereb.Blood Flow Metab. 15:929-40; Kumura, et al. (1996) Am. J. Physiol.270:C748-52) . The brain is very susceptible to oxidative stress-induceddamage because it is rich in polyunsaturated fatty acids and relativelylow levels of endogenous antioxidant enzymes in neuronal tissue toneutralize the effect of free radicals (Juurlink and Sweeney (1997)Neurosci. Biobehav. Rev. 21:121-8). It has been demonstrated that freeradicals and related reactive oxygen species mediate much of the damagethat occurs after transient brain ischemia (Love (1999) Brain Pathol.9:119-31). Moreover, SOD and glutathione peroxidase activities aresignificantly lower in stroke patients compared to control subjects,suggesting a significant correlation with infarct size, initial strokeseverity and poor short-term prognosis (Demirkaya, et al. (2001) Eur. J.Neurol. 8:43-51). These data suggest that superoxide anion, O₂ ⁻,hydrogen peroxide, and perioxynitrite are among the key reactive oxygenspecies implicated in ischemic injury.

Natural scavenger enzymes SOD and catalase are characterized by a veryhigh efficiency and great stability toward oxidants. Both enzymes havebeen used in their native form to prevent oxidative damage but they havebeen found to be effective only following repeated doses or whenadministered locally or in isolated tissues or cells. Further, targetingand unsatisfactory pharmacokinetics have limited the use of theseenzymes in methods of treatment (Abuchowski, et al. (1977) J. Biol.Chem. 252:3578-81; Monfardini and Veronese (1998) Bioconjug. Chem.9:418-50).

As an alternative, several synthetic, free radical scavengers have beenevaluated in animal models of cerebral ischemia and reperfusion and havebeen shown to be protective (Watanabe, et al. (1994) J. Pharmacol. Exp.Ther. 268:1597-604; Umemura, et al. (1994) Eur. J. Pharmacol. 251:69-74;Baker, et al. (1998) J. Pharmacol. Exp. Ther. 284:215-21; Itoh, et al.(1990) Psychopharmacology (Berl) 101:27-33; Gilgun-Sherki, et al. (2002)Pharmacol. Rev. 54:271-84). While some of the antioxidant-like SODmimetics show efficacy in animal models, poor stability of these agentsand the inability to sustain their retention at target sites are stillmajor challenges for their therapeutic use. Delivery of SOD to the brainhas been attempted via the use of prodrugs or carrier systems such asantibodies, liposomes (Kreuter (2001) Adv. Drug. Deliv. Rev. 47:65-81),and surface modifications such as conjugation to polyethylene glycol(SOD-PEG) (Veronese, et al. (2002) Adv. Drug Deliv. Rev. 54:587-606);however, treatment of cerebral ischemia/reperfusion injuries has beenlimited due to poor cerebral cell penetration. Gene therapy is analternative approach but the delivery of genes into brain tissues andexpression of therapeutic proteins may not be available for immediateeffect (Hermann, et al. (2001) Neurobiol. Dis. 8:655-66). Furthermore,cerebral protein synthesis is severely compromised in injured areasafter focal ischemia (Hermann, et al. (2001) supra; Hata, et al. (2000)J. Cereb. Blood Flow Metab. 20:937-46).

U.S. Pat. No. 6,123,956 teaches methods and compositions for treatingstroke and/or traumatic brain injury. The compositions taught in thisreference encompass a therapeutic agent such as SOD encapsulated in apharmaceutically acceptable polymer, e.g., polyesters such as PLA(poly(lactide)) and PLGA (poly(D,L-lactide-co-glycolide)), polyethyleneglycol, poloxomers, polyanhydrides, and pluronics), wherein thetherapeutic agent is present at therapeutically effective concentrationswhich, if injected into the cerebrospinal fluid of a subject sufferingfrom stroke or traumatic brain injury will contribute to theamelioration of the disorder.

SUMMARY OF THE INVENTION

The present invention relates to a method for inhibiting reperfusioninjury in the brain. The method involves administering an effectiveamount of an antioxidant, wherein said antioxidant is formulated in ananoparticle and administered via the carotid artery or jugular vein toa subject in need of treatment, thereby inhibiting reperfusion injury inthe brain of said subject. In certain embodiments, the antioxidant is anantioxidant enzyme (e.g., superoxide dismutase, catalase, glutathioneperoxidase, glutathione reductase, glutathione-S-transferasehemeoxygenase, or mimetic or synthetic enzymes thereof), a smallmolecule antioxidant (e.g., a vitamin antioxidant, acetyl salicyclicacid, mannitol, captopril, arginine, or pyruvate) or a combinationthereof. In other embodiments, the nanoparticle is composed of abiodegradable polymer such as a poly(lactide-co-glycolide), poly(lacticacid), poly(alkylene glycol), polybutylcyanoacrylate,poly(methylmethacrylate-co-methacrylic acid), poly-allylamine,polyanhydride, polyhydroxybutyric acid, or a polyorthoester or acombination thereof. In still further embodiments, the nanoparticlecontains a targeting moiety or a plasticizer such as L-tartaric aciddimethyl ester, triethyl citrate, or glyceryl triacetate to facilitatesustained release of the antioxidant.

The present invention further relates to a composition for sustainedrelease of an effective amount of an active agent. The compositioncontains an active agent (e.g., antioxidant, an anti-infective, anantiseptic, a steroid, a therapeutic peptide, an analgesic, ananti-inflammatory agent, an anticancer agent, a narcotic, an anesthetic,an antiangiogenic agent, a polysaccharide, a vaccine, an antigen, or anucleic acid), at least one biodegradable polymer (e.g., apoly(lactide-co-glycolide), poly(lactic acid), poly(alkylene glycol),polybutylcyanoacrylate, poly(methylmethacrylate-co-methacrylic acid),poly-allylamine, polyanhydride, polyhydroxybutyric acid, or apolyorthoester), and an inert plasticizer (e.g., L-tartaric aciddimethyl ester, triethyl citrate, or glyceryl triacetate). In particularembodiments, the nanoparticle composition further contains a targetingmoiety.

A method for effecting a sustained release of an effective amount of anactive agent using a nanoparticle containing an active agent, at leastone biodegradable polymer and an inert plasticizer is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows protein release of from nanoparticles containing dimethyltartrate (DMT). Release of bovine serum albumin (BSA) was determined ina double diffusion chamber separated by a permeable membrane which waspermeable to BSA but not nanoparticles. A suspension of nanoparticles (5mg/mL, 2.5 mL) was filled in the donor chamber and phosphate-bufferedsaline (PBS) (154 mM, pH 7.4, 37° C.) in the receiver chamber. Samplesfrom the receiver chamber were completely removed and replaced withfresh buffer. The protein levels in the released samples were analyzedby BCA protein assay. NP1, 90.0 mg polymer/0.0 mg DMT (0% DMT); NP2,85.5 mg polymer/4.5 mg DMT (5% DMT); NP3, 81.5 mg polymer/9.0 mg DMT(10% DMT); NP4, 63.0 mg polymer/27.0 mg DMT (30% DMT).

FIG. 2A shows that the addition of DMT to PLGA nanoparticles facilitatesthe release of DNA (PLGA 50:50, 143 kDa, 2% weight/volume PVA as anemulsifier). Nanoparticles were suspended in Tris-EDTA buffer andincubated at 37° C. The release DNA was separated by centrifugation andquantitated using Picogreen® (Promega, Madison, Wis.).

FIG. 2B shows the level of transfection of nucleic acid sequencesencoding luciferase when encapsulated in nanoparticles containing orlacking DMT. A single dose of nanoparticles (10 μg DNA) was used fortransfection of MCF-7 cells. The medium in the wells was changed on day2 and 4 after transfection with no addition of new nanoparticles.Luciferase levels were measured on day 3, 5, and 7 after transfection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for inhibiting reperfusioninjury in the brain using a highly effective course of therapy whichcombines an antioxidant formulated into a nanoparticle and injection ofthe nanoparticle formulation via the carotid artery or jugular vein.Using this protocol, it has now been shown that the nanoparticles cancross the blood brain barrier and inhibit ischemia in the brain. It hasfurther been demonstrated that when the nanoparticle formulationcontains an inert plasticizer such as dimethyl tartrate (DMT), sustainedrelease of the active agent can be achieved.

As it pertains to the present disclosure, ischemia is used in theclassical sense to refer to the condition suffered by tissues or organswhen deprived of blood flow; reduced blood flow results in an inadequatesupply of nutrients and oxygen in the tissues or organs. Reperfusioninjury refers to the tissue damage inflicted when blood flow is restoredafter an ischemic period of more than about ten minutes.

Antioxidants which can be formulated in a nanoparticle of the presentinvention to inhibit reperfusion injury include antioxidant enzymes,small molecule antioxidants, or combinations thereof. Antioxidants aresubstances which inhibit oxidation or suppress reactions promoted byreactive oxygen species such as oxygen itself, oxygen free radicals, orperoxides. Antioxidants can be absorbed into the cell membrane toneutralize oxygen radicals and thereby protect the membrane. As usedherein, antioxidant enzymes are generally proteins, or their fragments,that scavenge oxygen free radicals or H₂O₂ (hydrogen peroxide). Suitableantioxidant enzymes include, but are not limited to superoxidedismutase, catalase, glutathione peroxidase, glutathione reductase,glutathione-S-transferase or hemeoxygenase, or mimetic or syntheticenzymes thereof. See, U.S. Pat. No. 5,994,339 for mimetic enzymes.

Small molecule antioxidants include scavengers of .O₂ ⁻ (superoxide),.OH (hydroxyl) or NO (nitric oxide) radicals (e.g., acetyl salicylicacid, a scavenger of .O₂ ⁻; mannitol or captopril which are scavengersof .OH); molecules that inhibit the generation of these radicals (e.g.,arginine derivatives, inhibitors of nitric oxide synthase which produceNO; pyruvate which attenuates the rate of H₂O₂-induced generation ofreactive oxygen species); or vitamin antioxidants. Vitamin antioxidantsinclude lycopene; lutein; xeaxanthine; all forms of Vitamin A includingretinal and 3,4-didehydroretinal; all forms of carotene (e.g.,alpha-carotene, beta-carotene, gamma-carotene, delta-carotene); allforms of Vitamin C (e.g., D-ascorbic acid, L-ascorbic acid); all formsof Vitamin E such as tocopherol (e.g., alpha-tocopherol,beta-tocopherol, gamma-tocopherol, delta-tocopherol), tocoquinone,tocotrienol, and Vitamin E esters which readily undergo hydrolysis toVitamin E such as Vitamin E acetate and Vitamin E succinate, andpharmaceutically acceptable Vitamin E salts such as Vitamin E phosphate;prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E;pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, andVitamin E, and the like, and mixtures thereof. Analogues of Vitamin Esuch as TROLOX®, a compound which is more hydrosoluble than naturalforms of Vitamin E and which could reach intracellular sites morerapidly, is also contemplated.

Antioxidants for use in the formulations of the present invention can beisolated from a natural source or wholly or partially synthetically- orrecombinantly-produced. Methods for isolating or producing antioxidantsor antioxidant extracts are well-established in the art, see, e.g., U.S.Pat. Nos. 6,737,552; 6,660,320; 6,656,358; 6,653,530; 6,623,743;RE38,009; U.S. Pat. Nos. 6,429,356; 6,436,362; 6,262,279; 6,410,290;6,231,853; and 5,714,362 and WO 91/04315.

An effective amount of antioxidant present in a nanoparticle formulationof the present invention is an amount which prevents and/or reducesinjury of mammalian brain tissue due to ischemic conditions. Suchischemic conditions can arise from acute head trauma, surgical occlusionof blood flow, stroke, cardiac arrest and the like. The exact amount ofantioxidant will vary according to factors such as the antioxidant beingused as well as the other ingredients in the composition. Typically, theamount of antioxidant can vary from about 1 unit/kg to about 30,000units/kg of body weight or from about 500 units/kg to about 20,000units/kg. In particular embodiments, the antioxidant is given at a doseof about 10,000 units/kg. The effectiveness of the antioxidant treatmentcan be determined by monitoring the mammals neurological status, infarctvolume or plasma glucose levels as disclosed herein.

When the antioxidant is a mimetic, it has been demonstrated that the invivo oxidoreductase activity of the mimetic is such that an effectivedose will be low enough to avoid problems of toxicity (Faulkner, et al.(1994) J. Biol. Chem. 269:23471); therefore, doses that can be usedinclude those in the range of 1 to 50 mg/kg.

As disclosed herein, it has been found that an antioxidant-containingnanoparticle formulation can exert its effect via any route ofadministration; however, intracarotid administration is particularlyeffective at delivering a therapeutic amount of the active agent to thebrain. Accordingly, it is contemplated that an antioxidant-containingnanoparticle formulation of the present invention can be administeredvia intravenous, intracerebral, intracarotid, intramuscular orintrajugular routes, wherein intracarotid or intrajugular routes aresuitable. In particular embodiments, intracarotid administration isadvantageously used. The antioxidant-containing nanoparticle formulationof the present invention can be administered to a subject in need ofsuch treatment including a subject at risk of reperfusion injury (e.g.,in the case of surgery-induced ischemia) or a subject that hasexperienced an ischemic event (e.g., stroke) to prevent, inhibit and/orreduce reperfusion injury.

As one of skill in the art will appreciate, a nanoparticle in accordancewith the methods and compositions of the present invention can becomposed of a variety of injectable biodegradable polymers.Nanoparticles are said to be biodegradable if the polymer of thenanoparticle dissolves or degrades within a period that is acceptable inthe desired application (usually in vivo therapy), usually less thanfive years, and desirably less than one year, upon exposure to aphysiological solution of pH 6-8 having a temperature of between 25° C.and 37° C. As such, a nanoparticle for use in accordance with themethods and compositions of the present invention can be composedhomopolymers or copolymers prepared from monomers of polymers disclosedherein, wherein the copolymer can be of diblock, triblock, or multiblockstructure. Suitable polymers include, but are not limited to,poly(lactide-co-glycolides), poly(lactic acid), poly(alkylene glycol),polybutylcyanoacrylate, poly(methylmethacrylate-co-methacrylic acid),poly-allylamine, polyanhydride, polyhydroxybutyric acid, orpolyorthoesters and the like. In particular embodiments, a nanoparticleis composed of a copolymer of a poly(lactic acid) and apoly(lactide-co-glycolide). Particular combinations and ratios ofpolymers are well-known to the skilled artisan and anysuitable-combination can be used in the nanoparticle formulations of thepresent invention. Generally, the resulting nanoparticle typicallyranges in size from between 1 nm and 1000 nm, or more desirably between1 nm and 100 nm.

A nanoparticle of the present invention can further contain a polymerthat affects the charge or lipophilicity or hydrophilicity of theparticle. Any biocompatible hydrophilic polymer can be used for thispurpose, including but not limited to, poly(vinyl alcohol).

To further enhance delivery of a therapeutically effective amount of anactive agent, a nanoparticle of the present invention can furthercontain a targeting moiety (e.g., a protein transduction domain). Asused herein, a targeting moiety is any molecule which can be operablyattached to a nanoparticle of the present invention to facilitate,enhance, or increase the transport of the nanoparticle into targettissue. Such a moiety can be a protein, peptide or small molecule. Forexample, a variety of protein transduction domains, including the HIV-1Tat transcription factor, Drosophila Antennapedia transcription factor,as well as the herpes simplex virus VP22 protein have been shown tofacilitate transport of proteins into the cell (Wadia and Dowdy (2002)Curr. Opin. Biotechnol. 13:52-56). Further, an arginine-rich peptide(Futaki (2002) Int. J. Pharm. 245:1-7), a polylysine peptide containingTat PTD (Hashida, et al. (2004) Br. J. Cancer 90(6):1252-8), Pep-1(Deshayes, et al. (2004) Biochemistry 43(6):1449-57) or an HSP70 proteinor fragment thereof (WO 00/31113) is suitable for targeting ananoparticle of the present invention. Not to be bound by theory, it isbelieved that such transport domains are highly basic and appear tointeract strongly with the plasma membrane and subsequently enter cellsvia endocytosis (Wadia, et al. (2004) Nat. Med. 10:310-315). Animalmodel studies indicate that chimeric proteins containing a proteintransduction domain fused to a full-length protein or inhibitory peptidecan protect against ischemic brain injury and neuronal apoptosis;attenuate hypertension; prevent acute inflammatory responses; andregulate long-term spatial memory responses (Blum and Dash (2004) Learn.Mem. 11:239-243; May, et al. (2000) Science 289:1550-1554; Rey, et al.(2001) Circ. Res. 89:408-414; Denicourt and Dowdy (2003) TrendsPharmacol. Sci. 24:216-218).

Exemplary peptide-based targeting moieties are presented in Table 1.

TABLE 1 SEQ ID SOURCE AMINO ACID SEQUENCE NO: PTD-4^(a) YARAAARQARA 1HIV TAT^(a) YGRKKRRQRRR 2 PTD-3^(a) YARKARRQARR 3 PTD-5^(a) YARAARRAARR4 PTD-6^(a) YARAARRAARA 5 PTD-7^(a) YARRRRRRRRR 6 ANTp^(b)RQIKIWFQNRRMKWKK 7 Transportin^(b) GWTLNSAGYLLGKINLKALAALAKKIL 8 ^(a)Ho,et al. (2001) Cancer Res. 61:474-477. ^(b)Schwartz and Zhang (2000)Curr. Opin. Mol. Ther. 2:2.

Suitable small molecules targeting moieties which can be operablyattached to a nanoparticle of the present invention include, but are notlimited to, nonpeptidic polyguanidylated dendritic structures (Chung, etal. (2004) Biopolymers 76(1):83-96) or poly[N-(2-hydroxypropyl)methacrylamide] (Christie, et al. (2004) Biomed. Sci. Instrum.40:136-41).

To conjugate or operably attach the targeting moiety to a nanoparticleof the present invention, standard methods such as the epoxy activationmethod can be employed. The nanoparticle surface is contacted with anepoxy compound (e.g., DENACOL®, Nagase America Co., CA) which reactswith the hydroxyl functional group of, e.g., the PVA associated with thenanoparticle surface. The epoxy activation of the nanoparticle createsmultiple sites for reaction with a ligand and also serves as a linkagebetween the nanoparticle surface and the peptide to avoid sterichindrance for interaction of the peptide with the cell membrane(Labhasetwar, et al. (1998) J. Pharm. Sci. 87:1229-34). The epoxy groupscan react with many functional groups including amine, hydroxyl,carboxyl, aldehyde, and amide under suitable pH and buffer conditions;therefore increasing the number of possible targeting moieties which canbe employed.

A nanoparticle formulation of the present invention can further containa plasticizer to facilitate sustained release of the encapsulated activeagent by maintaining the structure of the nanoparticle. Release ofmolecules (e.g., proteins, DNA or oligonucleotides) from nanoparticlesformulated from block copolymers is, in general, not continuous.Typically, there is an initial release followed by a very slow andinsignificant release thereafter. Not to be bound by theory, it iscontemplated that the release profile may be as a result of the rapidinitial drop in the molecular weight of the polymer which reduces theglass transition temperature of the polymer to below body temperature(37° C.); the glass transition temperature of copolymers prior torelease is above body temperature (˜45 to 47° C.). Moreover, withdegradation, these polymers become softer thereby closing the poreswhich are created during the initial release phase (due to the releaseof active agent from the surface). Therefore, an inert plasticizer isadded to a nanoparticle formulation disclosed herein to maintain theglass transition temperature above 37° C. despite a decline in molecularweight of the polymer with time. In this manner, the pores remain openand facilitate a continuous release of the encapsulated active agent.Suitable plasticizers are generally inert and can be food/medical gradeor non-toxic plasticizers including, but not limited to, triethylcitrate (e.g., CITROFLEX®, Morflex Inc., Greensboro, N.C.), glyceryltriacetate (e.g., Triacetin, Eastman Chemical Company, Kingsport,Tenn.), L-tartaric acid dimethyl ester (i.e., dimethyl tartrate, DMT)and the like. A particularly suitable plasticizer is L-tartaric aciddimethyl ester.

The amount of plasticizer employed in a nanoparticle composition canrange from about 5 to 40 weight percent of the nanoparticle, moredesirably from about 10 to 20 weight percent of the nanoparticle. Inparticular embodiments, the plasticizer encompasses about 10 weightpercent of the nanoparticle composition.

By enhancing the release profile of an active agent, aplasticizer-containing nanoparticle has utility in the delivery of avariety of active agents to a variety of tissues or organs. Accordingly,the present invention further relates to a composition for sustained orcontinuous release of an effective amount of an active agent, whereinsaid composition contains an active agent, at least one biodegradablepolymer, and an inert plasticizer. As used herein, controlled release,sustained release, or similar terms are used to denote a mode of activeagent delivery that occurs when the active agent is released from thenanoparticle formulation at an ascertainable and controllable rate overa period of time, rather than dispersed immediately upon application orinjection. Controlled or sustained release can extend for hours, days ormonths, and can vary as a function of numerous factors. For thecomposition of the present invention, the rate of release will depend onthe type of the plasticizer selected and the concentration of theplasticizer in the composition. Another determinant of the rate ofrelease is the rate of hydrolysis of the linkages between and within thepolymers of the nanoparticle. Other factors determining the rate ofrelease of an active agent from the present composition include particlesize, acidity of the medium (either internal or external to the matrix)and physical and chemical properties of the active agent in the matrix.

In addition to delivery of antioxidants to the brain, a sustainedrelease nanoparticle formulation containing a plasticizer can be used todeliver any natural or synthetic, organic or inorganic molecule ormixture thereof in an amount which is sufficient to effect prevention ortreatment of a disease or condition in a subject. As used herein, anactive agent includes any compound or mixture of compounds whichproduces a beneficial or useful result. Active agents aredistinguishable from such components as vehicles, carriers, diluents,lubricants, binders and other formulating aids, and encapsulating orotherwise protective components. Examples of active agents arepharmaceutical, agricultural or cosmetic agents. Suitable pharmaceuticalagents include locally or systemically acting pharmaceutically activeagents which can be administered to a subject according to standardmethods of delivering nanoparticles (e.g., topical, intralesional,injection, such as subcutaneous, intradermal, intramuscular,intraocular, or intra-articular injection, and the like). Examples ofthese agents include, but not limited to, anti-infectives (includingantibiotics, antivirals, fungicides, scabicides or pediculicides),antiseptics (e.g., benzalkonium chloride, benzethonium chloride,chlorohexidine gluconate, mafenide acetate, methylbenzethonium chloride,nitrofurazone, nitromersol and the like), steroids (e.g., estrogens,progestins, androgens, adrenocorticoids, and the like), therapeuticpolypeptides (e.g. insulin, erythropoietin, morphogenic proteins such asbone morphogenic protein, and the like), analgesics andanti-inflammatory agents (e.g., aspirin, ibuprofen, naproxen, ketorolac,COX-1 inhibitors, COX-2 inhibitors, and the like), cancerchemotherapeutic agents (e.g., mechliorethamine, cyclophosphamide,fluorouracil, thioguanine, carmustine, lomustine, melphalan,chlorambucil, streptozocin, methotrexate, vincristine, bleomycin,vinblastine, vindesine, dactinomycin, daunorubicin, doxorubicin,tamoxifen, and the like), narcotics (e.g., morphine, meperidine,codeine, and the like), local anesthetics (e.g., the amide- oranilide-type local anesthetics such as bupivacaine, dibucaine,mepivacaine, procaine, lidocaine, tetracaine, and the like),antiangiogenic agents (e.g., combrestatin, contortrostatin, anti-VEGF,and the like), polysaccharides, vaccines, antigens, nucleic acids (e.g.,DNA and other polynucleotides, antisense oligonucleotides, and thelike), etc.

As will be appreciated by the skilled artisan, the nanoparticlecompositions of the present invention can further contain additionalfillers, excipients, binders and the like depending on, e.g., the routeof administration and the active agents used. A generally recognizedcompendium of such ingredients and methods for using the same isRemington: The Science and Practice of Pharmacy, Alfonso R. Gennaro,editor, 20th ed. Lippingcott Williams & Wilkins: Philadelphia, Pa.,2000.

By way of illustration, the compositions and methods of the presentinvention were employed in a rat model of reperfusion injury whereindelivery of the active agent was targeted to the brain. Localization ofDMT-containing nanoparticles in the brain, when administered viadifferent routes, was demonstrated using a formulation of nanoparticlesloaded with the fluorescent dye 6-coumarin (0.05%). In this manner, thedye acts as a marker and can be used to quantitatively determine theuptake of nanoparticles in cells or tissues (Panyam, et al. (2003) Int.J. Pharm. 262:1-11). Formulations containing rat serum albumin and 50 μgof dye were prepared as disclosed herein for BSA. The dye was dissolvedin the polymer solution prior to emulsification. A suspension ofnanoparticles in saline was infused (a 35 mg/kg dose dispersed in 500 μLof saline using water bath sonication) at the rate of 200 μL/minuteeither via the intracarotid, intrajugular vein, or intravenous route.These studies were carried out in animals in which no cerebral ischemiawas induced. One hour after nanoparticle administration, rats wereeuthanized, transcardially perfused with 200 mL of heparanized saline,and the brains collected for quantitative analysis of nanoparticleuptake. To analyze nanoparticle levels, brain samples were homogenizedin 100 μL saline, lyophilized for 48 hours, and the dry weight wasmeasured. To extract the dye from the nanoparticles localized in thetissue, 10 mL of methanol was added to each sample and incubated on anorbital shaker for 48 hours. After 48 hours, one milliliter of solutionwas taken from the tissue bottle and centrifuged at 14,000 rpm for 15minutes. The supernatant was collected and the dye concentration in thesample was determined using high performance liquid chromatography(HPLC). A standard plot using nanoparticles was prepared using identicalconditions to determine the amount of nanoparticles localized in thebrain.

The results of this analysis indicated that comparable uptake ofnanoparticles into the brain could be achieved via intravenous orintrajugular administration. With intracarotid administration, thenanoparticle brain levels were 20-fold higher (˜600 μg/gram of tissue)than that with intravenous or intrajugular administration. The brainuptake of DMT-containing nanoparticles via intracarotid route was about1.7% of the total dose that was administered, indicating that asignificant amount of DMT-containing nanoparticles can be localized tothe brain when administered via the intracarotid artery. Further, totalbrain uptake was independent of the condition of the brain as uptake ofdye-loaded nanoparticles following ischemia was found to be comparableto that in the normal brain via intravenous route of administration.

To demonstrate the effect of SOD on inhibition of ischemia in the brain,saline (n=4), SOD in solution (10,000 U/kg, n=2), low doseSOD-containing nanoparticles (10,000 U/kg, n=4) or high doseSOD-containing nanoparticles (20,000 U/kg, n=5) were administered torats via intracarotid route at the time of reperfusion. Thenanoparticles employed (40 mg/kg) were dispersed in 500 μL of saline andinfused via the carotid artery at the rate of 100 μL/minute. It wasfound that SOD in solution had no effect on infarct volume. Conversely,animals treated with SOD-containing nanoparticles exhibited asignificant 60% reduction in total infarct volume (low dose, ˜180 mm³;high dose, ˜130 mm³) as compared to that of saline control (˜345 mm³).

Behavioral data demonstrated that the motor and somatosensory functionswere impaired by the ischemic insult. Neurological deficit scores weresignificantly higher for animals administered saline control (deficitscore≈11) and SOD in solution (deficit score≈12) as compared to thatanimals administered SOD-containing nanoparticles (deficit score≈2-3).As animals receiving control nanoparticles demonstrated similar resultsas the saline control group, these data demonstrate that the beneficialoutcome imparted by SOD-containing nanoparticles was due to thesustained delivery of SOD to the brain by the nanoparticles.

The integrity of the blood-brain barrier was also assessed in animalsadministered SOD-containing nanoparticles. Cerebral ischemia wasdeveloped by occlusion of the middle cerebral artery for 60 minutes. Asolution of Evans blue dye (0.3 mL of 4% solution; Sigma, St. Louis,Mo.) was injected through the tail vein of the animals and immediately asuspension of SOD-containing nanoparticles (SOD dose=20,000 U/kg) wasinfused through the carotid artery prior to reperfusion. Six hours afterreperfusion, rats were sacrificed, transcardially perfused to removeblood and the brains were collected and photographed. A saline controlanimal showed intense coloration of the brain due to extravasation ofthe dye into the brain, indicating disruption of the blood-brainbarrier. In contrast, brains of animals infused with SOD-containingnanoparticles showed significantly lower extravasation of the dye. It isbelieved that the SOD-containing nanoparticles protected the endotheliumthereby maintaining the integrity of the blood-brain barrier andpreventing damage to the brain.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Formulation and Characterization of Nanoparticles

PLGA (90 mg; 50:50, inherent viscosity 1.31; Birmingham Polymers, Inc.,Birmingham, Ala.) was dissolved in 3 mL of chloroform. Dimethyl tartrate(DMT or tartaric acid dimethyl ester; density 1.238 g/mL; Sigma, St.Louis, Mo.) was dissolved in the polymer solution. Protein (30 mg ofBSA) was dissolved in 300 μL of water. The protein solution wasemulsified into the PLGA solution by vortexing for 1 minute and thensonicating for 2 minutes at 55 Watts energy output using a probesonicator (XL 2015 Sonicator® ultrasonic processor, Misonix, Inc.,Farmingdale, N.Y.).

The resulting primary emulsion was further emulsified into 12 mL of 2%PVA solution (PVA average molecular weight 30,000-70,000) by vortexingfollowed by sonicating for 2 minutes at 55 Watts. PVA solution wasfiltered through a 0.22 micron syringe filter and saturated withchloroform prior to use. A few drops of chloroform were added at a timeinto the PVA solution, shaken and the supernatant was used for theformulation.

The emulsion was stirred overnight on a stir plate at room temperaturefollowed by desiccation under vacuum for 1 hour. Nanoparticles thusformed were separated by centrifugation at 30,000 rpm for 30 minutes at4° C. (Beckman OPTIMA™ LE-80K, Beckman Instruments, Inc., Palo Alto,Calif.). Pelleted nanoparticles were resuspended in water andcentrifuged again as indicated above. The supernatant was collected andthe process was repeated one additional time to remove unencapsulatedprotein and emulsifier. The supernatants were collected and analyzed forprotein levels to determine the amount of protein not encapsulated inthe nanoparticles. Protein levels were determined using BIORAD® assaykit.

Nanoparticles were suspended in water by sonication as above. Thesuspension was lyophilized for 48 hours (VirTis Company, Inc. freezedryer, Gardiner, N.Y.).

The diameter of the nanoparticles was obtained with photon correlationspectroscopy (PCS) using quasi elastic light scattering equipment(ZETAPLUS™, zeta potential analyzer, Brookhaven Instruments Corp.,Holtsville, N.Y.) and ZETAPLUS™ particle sizing software (version 2.07).

To evaluate the release of nanoparticles containing DMT, DMT wasdissolved in the polymer solution, at various ratios, prior toemulsification of the model protein bovine serum albumin (BSA) or modelDNA sequence encoding luciferase.

It was found that with increasing concentrations of DMT, entrapmentefficiency of protein in the nanoparticles was reduced; however, theparticle size and polydispersity of nanoparticles was not significantlyaffected (Table 2).

TABLE 2 Polymer/DMT Protein Particle Size Polydispersity (mg) (% of DMT)Loading % (nm) Index 90.0/0.0 (0%) 18.3 396 0.12 85.5/4.5 (5%) 15.8 4040.13  81.5/9.0 (10%) 11.2 350 0.15 63.0/27.0 (30%) 10.8 326 0.14 *Meanhydrodynamic diameter measured by photon correlation spectroscopy in0.001 M HEPES buffer pH 7.0.

The release of BSA from nanoparticles containing 10% DMT was greaterthan that from nanoparticles lacking DMT (70% vs 30% in 30 days) (FIG.1). Further, the protein released from nanoparticles containing DMT hadsignificantly reduced aggregation as compared to that released fromnanoparticles lacking DMT.

Likewise, the release of DNA from nanoparticles containing 10% DMT wasenhanced compared to nanoparticles lacking DMT (FIG. 2A) and genetransfection was also facilitated (FIG. 2B). Addition of DMT at 10%concentration did not alter the DNA loading capacity or particle size ofthe nanoparticles compared to nanoparticles lacking DMT.

EXAMPLE 2 SOD-Containing Nanoparticles

SOD-containing nanoparticles with DMT were formulated as described forBSA. In general, 10% DMT was used in the nanoparticle formulations. Inaddition to SOD, rat serum albumin (RSA) was included (in the place ofBSA) and the SOD was dissolved into the RSA solution. Two formulationsof SOD-containing nanoparticles were prepared: low dose SOD-containingnanoparticles (˜10,000 Units SOD dose), 6 mg SOD (1 mg SOD=4,750 Units)and 24 mg RSA; and high dose SOD-containing nanoparticles (˜20,000 UnitsSOD dose), 12 mg SOD and 18 mg RSA.

SOD loading in nanoparticles was determined by analyzing the amount ofSOD that did not get encapsulated. For this purpose, the washingsgenerated during the preparation of the nanoparticles, as disclosedherein, were collected and analyzed for SOD enzyme activity using astandard SOD enzyme assay kit (Dojindo Labs, Kumamoto, Japan). SODloading into the nanoparticles was 4.5% (weight/weight; or 215 Units SODper milligram nanoparticles) with an encapsulation efficiency of 75%(i.e., 75% of the added protein was encapsulated into nanoparticles).

To demonstrate the sustained release nature of the nanoparticles, an invitro release study was carried out using a double diffusion chamberseparated by a hydrophilic low protein binding MILLIPORE® membrane (0.1μm porosity). The donor side of the diffusion cell was loaded with asuspension of nanoparticles containing 5 mg of nanoparticles in 2.5 mLphosphate-buffered saline (0.15 M, pH 7.4, 1% BSA, 0.05% TWEEN™ 20, and0.05% sodium azide at 37° C.). The receiver side contained the bufferdescribed above without nanoparticles. The solution from the receiverchamber was completely removed at regular time intervals and analyzedfor SOD levels.

The release study under in vitro conditions demonstrated sustainedrelease of the SOD protein encapsulated in nanoparticles. As the amountof SOD protein released was determined via enzyme activity, these dataindicate that the encapsulated and released SOD protein retained itsenzyme activity with time (Table 3).

TABLE 3 Time (Hours) % SOD Released 3 4.4 12 13 24 22.7 72 33.4 168 49.1

EXAMPLE 3 Targeting of Nanoparticles

Drug-loaded nanoparticles are surface modified with a proteintransduction domain such as TAT peptide (SEQ ID NO:2) using an epoxyactivation method. TAT peptide is a suitable targeting moiety as it hasbeen effectively conjugated to proteins and liposomes (Torchilin, et al.(2001) Proc. Natl. Acad. Sci. USA 98:8786-91). To conjugate or link theTAT peptide to a nanoparticle, the nanoparticle surface is contactedwith an epoxy compound (e.g., DENACOL®, Nagase America Co., Calif.)which reacts with the hydroxyl functional group of the PVA associatedwith the nanoparticle surface. In brief, 40 mg of drug-loadednanoparticle dispersed in 4 mL borate buffer (pH 5.0) and 12 mg DENACOL®524 (pentaepoxy) dissolved in equal volume of borate buffer is mixed andthe reaction is allowed to take place for 30 minutes at 37° C. withgentle stirring in the presence of zinc tetrafluroborate acting as acatalyst. The unreacted DENACOL® is separated from the nanoparticles byultracentrifugation followed by a single wash with borate buffer. Forconjugation to the TAT peptide, the epoxy-activated drug-loadednanoparticle is suspended in the borate buffer and mixed with a solutionof TAT peptide in 4 mL of borate buffer. The reaction is allowed to takeplace for 30 minutes at 37° C. and the unreacted peptide is separated byultracentrifugation with repeated washing with water.

The amount of TAT peptide can be optimized; however, a ratio of 1:10weight/weight TAT peptide to liposomes has been used to attachapproximately 100 to 500 molecules of TAT peptide per liposome particleof ˜200 nm in diameter (Torchilin, et al. (2001) supra). Further, usinga 40 nm diameter dextran-coated iron nanoparticle, it has beendemonstrated that as few as four TAT peptide molecules per particle wereeffective in achieving ˜200-fold greater particle uptake intohematopoietic and neural progenitor cells as compared to unmodifiedparticles (Lewin, et al. (2000) Nat. Biotechnol. 18:410-4). Therefore,based on the molecular weight of TAT peptide (1560 Da), an estimatedfour TAT peptide molecules per particle and the number of nanoparticleshaving a mean diameter of 25 nm present per mg (˜10¹²), it iscontemplated that 0.156 μg of peptide per mg weight of nanoparticles issuitable.

To determine the stability of the TAT peptide-nanoparticle conjugate,nanoparticles conjugated to FITC-labeled TAT peptide are dispersed in aserum-containing medium with aliquots centrifuged at different timepoints so that supernatants can be measured for fluorescence which isindicative of degradation of the nanoparticle.

EXAMPLE 4 Antioxidant Neuroprotection in Ischemia/Reperfusion Injury

Surgery leading to focal cerebral ischemia was performed under ketamine(80 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally andsupplemented as necessary during the procedure. Focal cerebral ischemiawas accomplished by means of a modification of the establishedintraluminal thread model (3-0 nylon monofilament suture) (Koizumi, etal. (1986) Jpn. J. Stroke 8:1-8). Briefly, the left common, internal,and external carotid arteries were exposed through a ventral midlineneck incision. The external carotid artery and common carotid arterywere ligated and a 19 to 22 mm length of poly-L-lysine-coated 3-0 nylonsuture was introduced into the lumen of the internal carotid artery andwas advanced to block the origin of the middle cerebral artery. After 60minutes of ischemia, the monofilament was removed, the wound wassutured, and the animal was allowed to recover from anesthesia with freeaccess to food and water. A subcutaneous injection of saline (2 mL) wasadministered to prevent post-anesthetic dehydration.

Neurological evaluations were carried out at six hours after theinduction of ischemia and reperfusion and the animals were subsequentlyeuthanized for histological analysis of the brain to determine theextent of focal ischemia. Neurological evaluations were performedaccording to a fourteen-point scale (Table 4).

TABLE 4 Maximum Motor Tests Points Muscle Status: Hemiplegia Raising therat by the tail Flexion of forelimb 1 Flexion of hindlimb 1 Head movingmore than 100 (vertical axis) 1 Placing the rat on the floor Inabilityto walk straight 1 Circling toward the paretic side 1 Falling down tothe paretic side 1 Abnormal movements Immobility and staring 1 Tremor(wet-dog shakers) 1 Myodystony, irritability, seizures 1 Sensory TestVisual and tactile placing 1 Proprioceptive test (deep sensory) 1Relexes (blunt or sharp stimulation) absent of: Pinna reflex (a headshake when touching 1 the auditory meauts) Corneal reflex (an eye blinkwhen lightly 1 touching the cornea with cotton) Startle reflex (a motorresponse to a 1 brief, loud paper noise) Maximum Points 14 (Longa, etal. (1989) Stroke 20: 84-91; Minematsu, et al. (1992) Stroke 23:1304-1311)

After ischemia/reperfusion and neurological score evaluation, rats weresacrificed with an overdose of sodium pentobarbitone and transcardiallyperfused with normal saline. Brains were carefully removed, andsectioned into six, 2 mm-thick coronial slices using rodent brainmatrices (Electron Microscopy Sciences, Hatfield, Pa.). Corneal brainslices were immediately immersed into 2% 2,3,5-triphenyltetrazoliumchloride (Sigma, St. Louis, Mo.) for 20 minutes at room temperature inthe dark followed by fixation in a 4% paraformaldehyde overnight.Infarct volume was calculated using the indirect method, in which theinfarcted area of the brain slice was first determined using the NIHImage program by subtracting the undamaged are of ipsilateral hemispherefrom the total of the contralateral hemisphere (Swanson, et al. (1990)J. Cereb. Blood Flow Metab. 10:290-3). The infarcted area was thenmultiplied by section thickness (2 mm) to obtain infarct volume for thatslice. Total brain infarct volume was finally obtained by summing thevolumes of the series of six brain slices prepared from each animal.

EXAMPLE 5 Additional Measurements for Assessing Ischemia/ReperfusionInjury

Intra-ischemic plasmic glucose is an important determinant of infarctsize (He, et al. (1993) supra). Plasma glucose levels are determinedusing any commercially available glucose analyzer. Arterial blood gasesand pH are measured using a Radiometer blood gas analyzer (Copenhagen,Denmark).

Local cortical blood flow (LCBF) is monitored in the left hemisphere inthe supply territory of the middle cerebral artery by laser Dopplerflowmetry. Each animal is placed supine, and the head is firmlyimmobilized in a stereotaxic frame (model 900, David Kopf Instruments) .Burr holes (1.5-mm diameter) are drilled 5-6 mm lateral and 1-2 mmposterior to bregma, without injury to the dura mater. The laser Dopplerflow probe is carefully positioned on the craniectomy site and LCBF iscontinuously monitored (2-Hz sampling rate) from before the onset ofischemia, during and five minutes after reperfusion. Flow values,averaged over 30-second periods, are collected every 10 minutes, withshorter intervals immediately after induction of ischemia andreperfusion. Decreased levels of LCBF, during intraluminal filamentinsertion, are expressed as a percentage of baseline flow (ischemicLCBF/preischemic LCBF)×100) . According to established methods (Imai, etal. (2002) Stroke 32:2149-54), if ischemic LCBF is not reduced withstabilization at <35% of the baseline signal, middle cerebral arteryocclusion is regarded as incomplete and animal are excluded from thestudy.

1. A method for inhibiting reperfusion injury in the brain comprisingadministering an effective amount of an antioxidant, wherein saidantioxidant is formulated in a biodegradable nanoparticle andadministered via the carotid artery to a subject having cerebralischemia, said nanoparticle providing sustained release of saidantioxidant, thereby inhibiting reperfusion injury in the brain of saidsubject.
 2. The method of claim 1, wherein the antioxidant comprises anantioxidant enzyme, small molecule antioxidant, or a combinationthereof.
 3. The method of claim 2, wherein the antioxidant enzymecomprises superoxide dismutase, catalase, glutathione peroxidase,glutathione reductase, glutathione-S-transferase hemeoxygenase, ormimetic or synthetic enzymes thereof.
 4. The method of claim 2, whereinthe small molecule antioxidant comprises a vitamin antioxidant, acetylsalicyclic acid, mannitol, captopril, arginine, or pyruvate.
 5. Themethod of claim 4, wherein the vitamin antioxidant comprises lycopene,lutein, xeaxanthine, Vitamin A, carotene, Vitamin C, tocopherol, orprodrugs or pharmaceutically acceptable salts thereof.
 6. The method ofclaim 1, wherein the nanoparticle comprises a biodegradable polymercomprising a poly(lactide-co-glycolide), poly(lactic acid),poly(alkylene glycol), polybutylcyanoacrylate,poly(methylmethacrylate-co-methacrylic acid), poly-allylamine,polyanhydride, polyhydroxybutyric acid, or a polyorthoester or acombination thereof.
 7. The method of claim 1, wherein the nanoparticlefurther comprises a targeting moiety.
 8. The method of claim 1, whereinthe nanoparticle further comprises a plasticizer to facilitate sustainedrelease of the antioxidant.
 9. The method of claim 8, wherein theplasticizer comprises L-tartaric acid dimethyl ester, triethyl citrate,or glyceryl triacetate.